Our previous SPORE project identified several genes silenced during the multistage pathogenesis of lung cancer, and methylation of six of these are being tested as intermediate markers of response in chemoprevention trials. In this proposal we will build on this theme, and we will study the clinico-pathological significance of evasion of apoptosis and tumor invasion, two of the hallmarks of lung cancer. Our proposal combines the cell biology/pathology expertise of Adi Gazdar with the apoptosis experience of Preet Chaudhary and will be supported by collaborators at the British Columbia Cancer Agency, Vancouver. Deregulation of apoptosis occurs at multiple levels of the death receptor and mitochondrial pathways Over-expression of the catalytically inactive caspase 8 homologue c-FLIP is not only anti-apoptotic, but also associated with drug resistance to lung cancer cells. Loss of cell adhesion cadherin molecules CDH1 and CDH13 and the crucial integrin-laminin combination (five independent genes) that anchors epithelial cells to the basement membrane are frequent in lung cancers. The latter is of special importance as it represents the very first step in tumor invasion. Deregulation of these genes occurs by aberrant methylation and by other mechanisms. After testing expression of over 50 molecules, we selected 20 for initial detailed study, using assays for methylation (methylation specific PCR and real time PCR) and protein expression (Western blots and immunostains), and another 10 molecules will be identified and studied during the course of this proposal. In Aim 1 we will identify 30 genes whose expression is deregulated by testing panels of lung cancer cell lines and tumors. In Aim 2 we will determine if aberrant methylation is the mechanism of silencing of these 30 genes and we will establish and validate methylation specific and real time PCR assays for them. Aim 3 will determine when deregulation of these molecules occurs during the multistage pathogenesis of lung cancer, so as to determine whether they can be used as intermediate markers for chemoprevention studies. In Aim 4 we will study 300 well-characterized resected NSCLCs to determine if deregulated expression of these molecules, either individually or in combination, is correlated with survival or multiple other clinico-pathological features. We will utilize resources from all of the Cores and interact with two other projects. Translational aspects include identification of markers for prognosis, chemoprevention and risk assessment. Our studies will greatly aid the understanding and clinical application of two crucial hallmarks of lung cancer.